A traditionally successful approach to viral vaccines, whole inactivated virus (WIV), has received relatively little attention in the overall effort to develop an effective HIV/AIDS vaccine. Only one inactivated virus product (the Salk/IRC Immunogen, "Remune") has been tested in humans but this product is envelope depleted and tested only as a therapeutic rather than a prophylactic immunogen. [unreadable] Since the primary target epitopes for antibody neutralization are in the envelope glycoprotein, it is generally considered an indespensible component of a prophylactic immunogen. Thus far, however, immunization of humans with monomeric recombinant envelope glycoprotein (rgp120) has resulted in relatively type-specific neutralization, very little neutralization of primary isolates, and no efficacy. The few monoclonal antibodies that demonstrate broad primary-isolate neutralization generally recognize conformational epitopes. The primary hypothesis driving this proposal is that such epitopes can be retained, and presented in an immunogenic form, on WIV. [unreadable] Our prior work focused on 1) developing methods for scalable propagation of diverse primary isolates of HIV-1, 2) assessing the retention and structure/function of gp120 during various methods for propagation, processing, and inactivation, 3) evaluating the combination of two orthogonal inactivation procedures, and 4) assessing the animal immunogenicity of WIV, using one set of possible production protocols and vaccine formulation. Preliminary studies were promising, showing relatively broad heterologous neutralization of primary isolates, despite rather small doses and low purity of the WIV. The objective of this proposal is to evaluate promising but unproven methods for virus stabilization, to achieve dramatic improvements in the yield, purity, and immunogenicity of WIV without sacrificing the structural integrity of the native gp120 trimers. These novel methods for virus stabilization are based on studies of cryptobiotic species that are capable of surviving drastic but transient environmental conditions (e.g. high salt, high heat, freezing, and desiccation) due to high concentrations of solutes, which are variably known as osmolytes, kosmotropes, or compatible solutes. The best studied of these is the disaccharide trehalose, which has been used to stabilize viral vaccines during lyophylization. [unreadable] We hypothesize that the innovative use of such solutes will yield dramatically improved WIV immunogenicity as well as the scalability that will be needed if this candidate were to proceed to human testing. In addition to the benefits of this approach for the WIV vaccine concept, methods for improving the purity and yield of intact viruses during processing and purification could contribute to many other HIV vaccine concepts (e.g. those using recombinant viral vectors such as vaccinia, avipox, VSV, VEE, etc.) and could also impact the gene therapy field where retroviruses are commonly used as the delivery vehicle and product yield is a major limiting factor. [unreadable] If this effort is successful at improving WIV yield and immunogenicity (R21 phase), we will conduct a proof-of-principle efficacy study in macaques using subtype-C WIV as the vaccine, followed by challenge with a heterologous subtype C infectious and pathogenic SHIV (R33 phase). Therefore, this proposal represents a critical step in qualifying this HIV vaccine concept for transition from pre-clinical to clinical studies. [unreadable] [unreadable] [unreadable]